Filovirus vectors and noninfectious filovirus-based particles

ABSTRACT

Cloned filovirus genomic cDNA and methods of using the cDNA are provided. Further provided are noninfectious lipid encapsulated filovirus-based particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/353,856, filed Jan. 29, 2003, which claims the benefit of the filingdate of U.S. application Ser. No. 60/353,972, filed on Jan. 31, 2002,under 35 U.S.C. §119(e). Which applications are incorporated byreference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support awarded by the NationalInstitutes of Health, Grant Nos. AI42774 and AI44386. The Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

Ebola virus, a member of the family Filoviridae and the orderMononegavirales, is an enveloped, nonsegmented negative-strand RNA virusand is one of the most lethal human and nonhuman primate pathogensrecognized to date (Feldmann et al., 1998; Vanderzanden et al., 1998).Four subtypes of Ebola virus have been identified, including Zaire,Sudan, Ivory Coast, and Reston (Sanchez et al., 1993). Human infectionwith subtype Zaire causes a fulminating, febrile, hemorrhagic diseasethat results in extensive mortality (Feldmann et al., 1993). Thus, Ebolavirus infection presents a much-needed model to study virus-inducedmechanisms leading to coagulation disorders and vascular instability.However, identification of major determinants of Ebola viruspathogenicity has been hampered by the lack of effective strategies forexperimental mutagenesis.

Ebola virus particles have a filamentous appearance, but its shape maybe branched, circular, U- or 6-shaped, or long and straight (Feldmann etal., 1996). Virions show a uniform diameter of approximately 80 nm, butvary greatly in length. Ebola virus particles consist of sevenstructural proteins. The glycoprotein (GP) of Ebola virus forms spikesof approximately 7 nm, which are spaced at 5- to 10-nm intervals on thevirion surface (Feldmann et al., 1996 and Peters et al., 1995). Cleavageof the GP is thought to be an important determinant of viralpathogenicity (Volchkov et al., 1998; Sanchez et al., 1996; Takada etal., 1997; Volchkov et al., 1998a; Volchkov et al., 1998b; Wool-Lewis etal., 1998; Yang et al., 2000). The Ebola virus GP contains a highlyconserved consensus motif for the subtilisin-like endoprotease furin,and previous studies demonstrated GP cleavage by this protease (Nina etal., 1991). Nonetheless, studies of murine leukemia virus (Wood-Lewis etal., 1999) or vesicular stomatitis virus (VSV) (Ito et al., 2001)pseudotyped with mutant Ebola virus GPs lacking the furin recognitionmotif at the cleavage site, showed that GP cleavage by furin was notessential for infectivity of the pseudotyped viruses. In many viruses,GP cleavage by furin and related endoproteases is essential for theirinfectivity. Thus, the significance of GP cleavage for the Ebola viruslife cycle remains in question.

GP is the only transmembrane protein of Ebola virus, and is responsiblefor receptor binding and membrane fusion (Takada et al., 1997). Cellsinfected with recombinant vaccinia virus expressing the GP producedvirosomes that varied in shape and diameter but uniformly possessedspike structures on their surface (Volchkov et al., 1998c), although theeffects of over 80 vaccinia viral proteins (Moss, 1995) on the formationof particles are unknown. Similar virosomes are also released from Ebolavirus-infected cells (Volchkov et al., 1998c). These findings suggestthat the GP contributes not only to an early stage of the viralinfection cycle but also to viral budding.

In addition, although recent studies have begun to address the immuneresponse to viral infection (Baize et al., 1999; Basler et al., 2000;Vanderzanden et al., 1998; and Wilson et al., 2000), as well as thefunctions of the viral proteins involved in the replicative process(VP30, VP35, NP, L) (Basler et al., 2000 and Muhlberger et al., 1999)and GP, little is known about the functions of the viral proteinsassociated with the membrane, including viral protein 40 (VP40), whichappears equivalent to matrix protein of other viruses.

The matrix proteins of many nonsegmented, negative-sense RNA virusesplay a critical role in viral particle formation (virus assembly) andbudding (Garoff et al., 1998). Expression of the matrix protein of VSVin insect and mammalian cells results in evagination of matrixprotein-containing vesicles from the plasma membrane surface (Justice etal., 1995; and Li et al., 1993). Matrix proteins interact with membranesin a hydrophobic and/or electrostatic manner and electron micrographs ofnonsegmented, negative-sense RNA viruses have demonstrated that thematrix protein forms a layer associated with the inner leaflet of thelipid bilayer (Garoff et al., 1998).

VP40 is the most abundant protein in virions (it represents 38% of theprotein in the viral particle) and is located beneath the viralmembrane, where is presumably maintains the structural integrity of theparticle (Feldmann et al., 1996). VP40 is encoded by the third gene inthe linear 3′-5′ RNA genome of Ebola virus and is 326 amino acids inlength, which includes a number of hydrophobic regions (Elliott et al.,1985 and Sanchez et al., 1996). VP40 contains a PPXY motif (X denotesany amino acid) at amino acids 10-13 (Harty et al., 1996) that is alsopresent at amino acids 16-19 in Marburg virus, strain Popp (Sanchez etal., 1993). This motif has been shown to play an important role in thebudding of rabies virus and VSV: when either of the prolines or thetyrosine of this motif is altered in the matrix proteins of theseviruses, viral budding is markedly reduced by comparison to findingswith wild-type virus (Harty et al., 1996). Mutation of the PPXY motif inthe matrix protein of VSV appears to reduce virus yield by pre-emptingbudding of assembled virions at the plasma membrane (Jayakar et al.,2000). This motif interacts with the WW domain found in many cellularregulatory and signal transduction proteins (Bork et al., 1994 and Chenet al., 1995) and interactions between one or more cellular proteins andthe matrix proteins of these viruses are thought to be crucial forefficient virus release from cells (Harty et al., 1999).

The matrix proteins of many enveloped viruses are thought to interactwith the cytoplasmic tails of viral glycoproteins. Such interaction isbelieved to be important for virus assembly. In influenza viruses, theremoval of the cytoplasmic tail of the hemagglutinin or neuraminidaseglycoprotein alters virion morphology (Jin et al., 1997; Mitnaul et al.,1996). Although not essential for normal particle formation in rabiesvirus and VSV, glycoproteins enhance the efficiency of particleformation (Mebatsion et al., 1996; Mebatsion et al., 1999; Schnell etal., 1998).

Thus, what is needed is a method to readily manipulate the filovirusgenome.

SUMMARY OF THE INVENTION

The invention provides methods to prepare filovirus, e.g., Marburg virusand Ebola virus, from cloned DNA and compositions useful therefor. Asdescribed herein, a reverse genetics system was employed to generatefilovirus, e.g., Ebola virus, from cloned cDNA. The genomic sequence wasprepared by reverse transcription and amplification of viral RNA. Theexpression of the resulting genomic cDNA, e.g., in host cells, in senseand antisense orientation yields cRNA or vRNA, which in the presence ofcertain viral proteins, e.g., L, NP, VP30 and VP35, yielded infectiousvirus. This system was also used to generate a mutant virus with analtered furin cleavage motif in GP. When expressed in cells, the GP ofthe wild-type, but not of the mutant, virus was cleaved into GP1 andGP2. Although posttranslational furin-mediated cleavage of GP wasthought to be an essential step in Ebola virus infection, generation ofa viable mutant Ebola virus lacking a furin recognition motif in the GPcleavage site demonstrated that GP cleavage is not essential forreplication of Ebola virus in cell culture.

Thus, the invention provides a composition comprising a plurality offilovirus vectors. The composition comprises a vector comprising apromoter operably linked to a nucleic acid molecule comprising afilovirus genomic cDNA linked to a transcription termination sequence, avector comprising a promoter operably linked to a nucleic acid molecule,for instance, a DNA segment, encoding a filovirus RNAtranscriptase-polymerase, a vector comprising a promoter operably linkedto a nucleic acid molecule encoding a filovirus NP, a vector comprisinga promoter operably linked to a nucleic acid molecule encoding filovirusVP30, and a vector comprising a promoter operably linked to a nucleicacid molecule encoding filovirus VP35. Preferred promoters for thevector comprising the filovirus cDNA include, but are not limited to, aRNA polymerase I promoter, RNA polymerase II promoter, RNA polymeraseIII promoter, T7 RNA polymerase promoter, or T3 RNA polymerase promoter,and preferred transcription termination sequences include, but are notlimited to, a RNA polymerase I transcription termination sequence, RNApolymerase II transcription termination sequence, RNA polymerase IIItranscription termination sequence, or a ribozyme. The sequence of thefilovirus genomic cDNA may be that of wild-type or may have one or morenucleotide deletions, insertions or substitutions relative to thegenomic sequence of a corresponding wild-type filovirus. Virus, eitherwild-type or mutant, such as a randomly mutagenized sequence or onesubjected to directed evolution, prepared from such a cDNA, is useful toscreen for antiviral compounds or other desirable properties such asimmunogenicity, to prepare a vaccine which results in a protectiveimmune response when administered to animals, e.g., mammals andpreferably primates, or to deliver a nucleic acid sequence of interestto cells, e.g., a marker gene, a gene encoding an immunogenic proteinfrom a pathogen including viruses other than a filovirus, bacteria,fungi or yeast, or a therapeutic protein, e.g., ADA, CFTR, factor VIIIor factor IX. Further, as the length of a filovirus virion is variable,the nucleic acid sequence of interest may be introduced into clonedfilovirus cDNA, as an individual open reading frame, e.g., one encodinga functional protein, or so as to encode a fusion protein with afilovirus protein, or as a replacement (substitution) for one or morecoding regions in the filovirus genome. Depending on whether or notvirus replication is desirable, a filovirus cDNA which lacks one or morefilovirus coding regions but comprises a DNA of interest may beintroduced into a cell along with the full-length (genomic) cDNA,optionally with vectors encoding filovirus proteins. In particular, eachof the coding regions for genes not associated with filovirusreplication, e.g., GP, VP40 and VP24, may be replaced with a DNA ofinterest. The resulting virus-like particles may be employed to screenfor compounds with desirable pharmacological profiles, e.g., antiviralcompounds. Alternatively, a filovirus cDNA which lacks one or more viralcoding regions, but includes filovirus sequences for encapsidationand/or replication and includes a DNA of interest, may be introducedinto a cell along with vectors encoding filovirus proteins, to formvirus-like particles.

The invention thus also provides a method to prepare filovirus. Themethod comprises contacting a cell with a vector comprising a promoteroperably linked to a filovirus genomic cDNA or a portion thereof, e.g.,a portion which, when expressed as vRNA is packaged into virions and canbe replicated in the presence of filovirus proteins, linked to atranscription termination sequence, a vector comprising a promoteroperably linked to a nucleic acid molecule, e.g., a DNA segment,encoding a filovirus RNA transcriptase-polymerase, a vector comprising apromoter operably linked to a nucleic acid molecule encoding filovirusNP, a vector comprising a promoter operably linked to a nucleic acidmolecule encoding filovirus VP30, and a vector comprising a promoteroperably linked to a nucleic acid molecule encoding filovirus VP35, soas to yield infectious filovirus. A portion of a filovirus cDNA includesportions which, when transcribed, yield a RNA which is capable of beingpackaged into filovirus virions or which is capable of being replicatedin the presence of filovirus proteins. In one embodiment, the genomiccDNA may have been recombinantly manipulated, for example, byintroducing one or more nucleotide deletions, insertions orsubstitutions. The promoters may be recognized by RNA polymerasesexpressed in the cells to be transfected, transformed or transduced withthe vectors of the invention, or may be recognized by a RNA polymerasethat is introduced to the cell concurrently or sequentially with thefiloviral vectors, e.g., by introduction of the polymerase itself or avector encoding the polymerase. In one embodiment, the filovirus genomiccDNA may be manipulated to encode a fusion protein, encode a therapeuticprotein or a protein useful in a vaccine, e.g., an immunogenictumor-specific protein or an immunogenic peptide or protein of apathogen, such as a bacteria, virus, yeast, or fungus. Also provided arecells contacted sequentially or concurrently with a composition, vectoror virus of the invention, virus obtained by the methods of theinvention, and cells infected with the virus.

As also described herein, VP40, when expressed apart from other viralproteins in mammalian cells, induced particle formation, which differedin length but with uniform diameters of approximately 65 nm. Efficientparticle formation may rely on a conserved N-terminal PPXY motif, asmutation or loss of this motif resulted in markedly reduced particleformation. These findings demonstrate that VP40 alone possesses theinformation necessary to induce particle formation, and this processmost likely requires cellular WW-domain-containing proteins thatinteract with the PPXY motif of VP40. Flotation gradient analysisindicated that VP40 binds to membranes in a hydrophobic manner, as NaClat 1 M did not release the protein from the lipid bilayer. Triton X-114phase-partitioning analysis suggested that VP40 possesses only minorfeatures of an integral membrane protein. Truncation of the C-terminal50 amino acids of VP40 resulted in decreased association with cellularmembranes, and demonstrated that this deletion disrupts hydrophobicinteractions of VP40 with the lipid bilayer, as well as abolishingparticle formation. Truncation of the C-terminal 150 amino acids orN-terminal 100 amino acids of VP40 enhanced the protein's hydrophobicassociation with cellular membranes. These mutants may be useful asdominant negatives, to determine targets for antivirals.

When the Ebola virus GP was expressed in cells, pleomorphic particleswere found budding from the plasma membrane. By contrast, when GP wasco-expressed with VP40, GP was found on the filamentous particlesinduced by VP40. These results demonstrated the central role of VP40 inthe formation of the filamentous structure of Ebola virions and suggestsan interaction between VP40 and GP in morphogenesis.

Thus, the invention provides a method to prepare lipid encapsulatedparticles comprising recombinant filovirus matrix protein. The methodcomprises providing a culture of eukaryotic cells contacted with avector comprising a promoter operably linked to a nucleic acid, e.g.,DNA, encoding a filovirus matrix protein or a portion thereof which iscapable of being incorporated into a filovirus particle. Supernatantfrom the culture which comprises lipid encapsulated particles comprisingfilovirus matrix protein is then collected. Preferred eukaryotic cellsare mammalian cells, including primate cells such as monkey or humancells, although any eukaryotic cell, in which the expression of VP40results in VP40-containing particles in supernatants, may be employed.The particles prepared by the method are useful as nucleic acid (DNA orRNA) or protein delivery vehicles, e.g., as replication incompetentvirus-like particles useful as a vaccine or a tolerogen, e.g., tosuppress or inhibit an immune response to an endogenous antigen, e.g.,myelin basic protein, collagen, thyroglobulin, acetylcholine receptor,DNA, or islet cell antigens, or an exogenous antigen, e.g., proteinantigens of Alternaria alternata (Alt a I), Artemisia vulgaris (Art vII), Aspergillus fumigatus (Asp f II), Dermatophagoides pteron. (Der pI, Der pIII, Der p IV, Der p VI and Der p and domestic animals such asFelis domesticus (Fel d I), cows, pigs, poultry, mice, hamsters,rabbits, rats, guinea pigs, dogs and horses. Common fungal antigensinclude those of Basidiomycetes such as Ustilago, Ganoderma, Alternaria,Cladosporium, Aspergillus, Sporobolomyces, Penicillium, Epicoccum,Fusarium, Phoma, Borrytis, Helminthosporium, Stemphylium andCephalosporium; Phycomycetes such as Mucor and Rhizopus; and Ascomycetessuch Eurotium and Chaetomium.

Accordingly, the eukaryotic cell may also express a nucleic acid, e.g.,DNA, fragment of interest, including, but not limited to, one whichencodes a therapeutic protein or peptide, an immunogenic peptide orprotein of a pathogen, a tumor antigen or an immunogenic peptidethereof, a transmembrane protein such as one which specifically binds toa receptor on a particular cell type or tissue, a viral glycoproteinwhich specifically binds to a receptor on a particular cell type ortissue, or a fusion thereof with a filovirus GP. In one embodiment,cells express filovirus matrix protein and a fusion (chimera) of afilovirus glycoprotein, e.g., the transmembrane domain and intracellulardomain of the filovirus glycoprotein and the extracellular portion of anon-filovirus protein, e.g., the extracellular domain of a cellular orviral transmembrane protein, such as influenza virus HA, or a solublepeptide or protein (one which does not comprise a transmembrane domain)which binds to a particular receptor. The resulting lipid encapsulatedparticles specifically bind to cells having a receptor for theextracellular non-filovirus protein or the soluble peptide or protein.In this manner, the lipid encapsulated particles may be targeted to aspecific cell type or tissue in an animal and can deliver theencapsulated content(s) of the particle to the specific cell type ortissue. Also provided are isolated and/or purified lipid encapsulatedparticles obtained by the method. Such particles may be employed toscreen for antiviral compounds, e.g., antivirals for other nonsegmentedviruses.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Generation of Ebola virus entirely from cloned cDNA. (A)Schematic diagram of cDNA plasmids for Ebola virus cRNA or (B) vRNAsynthesis and their efficiencies for virus generation. T7 and Ribindicate T7 RNA polymerase promoter and ribozyme sequences,respectively. G designates a guanine nucleotide inserted between thepromoter and Ebola virus cDNA. Synthesis of positive-sense Ebola viruscRNA is represented by “Ebola,” while the inverse lettering denotessynthesis of negative-sense vRNA.

FIG. 2. Replication of Ebola virus in cell culture. A) Six days afterinfection (light microscope). B) Three days after infection(antibody-based staining for virus). Mutant refers to an Ebola viruswith an altered furin recognition sequence in GP.

FIG. 3. Replication kinetics of wild-type Ebola virus and its GPcleavage mutant.

FIG. 4. Comparison of GP cleavage between wild-type and mutant Ebolavirus. Labeled proteins were separated on 8% (A) and 15% (B) sodiumdodecyl sulfate-polyacrylamide gels under reducing conditions. M,molecular mass marker; lane 1, mock-infected Vero E6 cells; lane 2,wild-type Ebola virus generated from plasmids; lane 3, GP cleavagemutant virus.

FIG. 5. (A) Schematic representation of wild-type VP40 and VP40 mutants.Substituted residues are indicated in bold-face type. (B) Kyte-Doolittlehydrophobicity plot of Ebola virus VP40 over a window of 17 amino acids(Justice et al., 1995).

FIG. 6. Expression of VP40, VP40/M14A and VP40AAXY in 293T cells. Thesample for the negative control was prepared from cells transfected withthe empty vector (pCAGGS/MCS). Lysates were harvested 24 hourspost-transfection, and proteins were separated by SDS-PAGE (12%) anddetected by Western blotting.

FIG. 7. Particle formation by VP40 and its mutants. Lanes representfractions from a sucrose gradient (numbered from the top) loaded withVP40 (A) or a mutant VP40(B-D) from cells transfected with VP40-encodingconstructs. Proteins were separated by SDS-PAGE (12%) and detected byWestern blotting.

FIG. 8. Protease protection analysis of VP40-induced particles. Lane 1:no treatment; lane 2: soybean trypsin inhibitor; lane 3: Triton X-100;lane 4: trypsin; lane 5: Triton X-100 and trypsin; and lane 6: trypsininhibitor and trypsin. Proteins were separated by SDS-PAGE (12%) anddetected by Western blotting.

FIG. 9. Membrane-association analysis of VP40 and its deletion mutants.Shown are gradients from cells expressing VP40 (A-B), VP40/1-276 (C-D),VP40/1-226 (E-F), VP40/1-176 (G-H), VP40/50-326 (1-J), and VP40/100-326(K-L). Fractions are numbered from the top to the bottom of thegradient. Proteins were separated by SDS-PAGE (12%) and detected byWestern blotting.

FIG. 10. Membrane-association analysis of VP40 mutants VP40AAXY (A) andVP40M14A (B). Lanes represent fractions collected from the top of agradient formed with a homogenate in 80% sucrose overlaid with 65% and10% sucrose layers. Lanes represent fractions collected from the top ofa gradient formed with a homogenate in 80% sucrose overlaid with 65% and10% sucrose layers. Proteins were separated by SDS-PAGE (12%) anddetected by Western blotting.

FIGS. 11A-G. Triton X-114 phase partitioning analysis of VP40 and itsdeletion mutants. The homogenate was partitioned into aqueous (A) anddetergent (D) phases. Proteins were separated by SDS-PAGE (12%) anddetected by Western blotting.

FIG. 12. Budding of GP-associated particles from the plasma membrane.Twenty four hours post-transfection of 293T cells with a GP-expressingplasmid (A). 293T cells transfected with an empty expression vector lacksuch particle formation (B). Bar, 100 nm.

FIG. 13. Pleomorphic particles resulting from GP expression. Thesupernatants of cells expressing GP were centrifuged through 20%sucrose, and the pelleted material was then negatively stained with 2%PTA. Pleomorphic particles with surface spikes were observed (A and B).Pelleted material was also immunolabeled with a mixture of anti-GPmonoclonal antibodies conjugated to 15-nm gold particles C) and D). Bar,100 nm.

FIG. 14. Morphologic changes in 293T cells expressing VP40. At 24 hpost-transfection of 293T cells with a VP40-expressing plasmid,filamentous particles budding from the plasma membrane (A), membraneruffles and the adhering site of two bilayers (C, arrows), as well asaggregated ribosomes (E, arrows) were apparent. Intracellularelectron-dense filamentous structures (F, arrowheads) were alsoobserved. The filamentous particles and membrane ruffles wereimmunolabeled with an anti-VP40 antibody conjugated with 5-nm goldparticles (B and D). M, mitochondrion; mt, microtubule. Bar, 100 nm (A,B, C, D, F) or 200 nm (E).

FIG. 15. Filamentous particles induced by VP40 expression. Thesupernatants of cells expressing VP40 were centrifuged through 20%sucrose, and the pelleted material was then negatively stained with 2%PTA. Particles with uniform diameters of approximately 65 nm and variedlengths were observed (A-C). Bar, 100 nm.

FIG. 16. Filamentous, spiked particles budding from the plasma membrane24 hours after-transfection of 293T cells with plasmids coexpressingVP40 and GP (A and B). Bar, 100 nm.

FIG. 17. Ebola virus-like particles produced by coexpression of VP40 andGP. The supernatants of cells coexpressing these two proteins werecentrifuged through 20% sucrose, and the pelleted material was thennegatively stained with 2% PTA. Filamentous particles with surfacespikes and varied lengths were observed (A-C). Pelleted material wasalso immunolabeled with a mixture of anti-GP monoclonal antibodiesconjugated to 15-nm gold particles (D, arrowheads), or treated with0.03% Triton X-100 at room temperature for 15 minutes, and thenimmunolabeled with a mixture of anti-GP antibodies conjugated to 15-nmgold particles (E, arrowheads) and an anti-VP40 antibody conjugated to5-nm gold particles (E, arrows). Bar, 1 μm (A) or 100 nm (B-E).

FIGS. 18A-18KKK. Representative filovirus sequences (Accession numbersAB050936, NC002549, NC001608, AF086833 and AF272001).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides isolated and/or purified vectors or plasmids,which encode filovirus proteins, and/or express filovirus genomic RNA.When introduced into a cell, these vectors yield infectious filovirus.Thus, the invention includes isolated and/or purified filovirus preparedby the methods disclosed herein. As also described, the inventionprovides isolated and/or purified noninfectious lipid encapsulatedparticles, i.e., the contacting of cells with noninfectious particlesdoes not yield progeny virus. As used herein, the terms “isolated and/orpurified” refer to in vitro preparation, isolation and/or purificationof a vector, plasmid, virus or lipid encapsulated particle of theinvention, so that it is not associated with in vivo substances, or issubstantially purified from in vitro substances. As used herein, theterm “recombinant nucleic acid” or “recombinant DNA sequence, fragmentor segment” refers to a nucleic acid, e.g., to DNA, that has beenderived or isolated from a source, that may be subsequently chemicallyaltered in vitro, and includes, but is not limited to, a sequence thatis naturally occurring, is not naturally occurring, or corresponds tonaturally occurring sequences that are not positioned as they would bepositioned in the native genome. An example of DNA “derived” from asource, would be a DNA sequence that is identified as a useful fragment,and which is then chemically synthesized in essentially pure form. Anexample of such DNA “isolated” from a source would be a useful DNAsequence that is excised or removed from said source by chemical means,e.g., by the use of restriction endonucleases, so that it can be furthermanipulated, e.g., amplified, for use in the invention, by themethodology of genetic engineering.

The vectors or plasmids of the invention comprise filovirus cDNA, forexample, one or more open reading frames encoding filovirus proteins orportions of the genomic sequence which are capable of being replicatedand packaged into virions in the presence of filovirus proteins.Therefore, gene(s) or portions thereof other than those of a filovirusmay be employed in the vectors or plasmids, or methods, of theinvention. A vector or plasmid of the invention may comprise a gene oropen reading frame of interest, e.g., a foreign gene encoding animmunogenic peptide or protein useful as a vaccine or a therapeuticprotein. If more than one vector is employed, the vectors may bephysically linked or each vector may be present on an individual plasmidor other, e.g., linear, nucleic acid delivery vehicle. The vectors orplasmids may be introduced to any host cell, preferably a eukaryoticcell. Preferred host cells to prepare virus or lipid encapsulatedparticles of the invention include insect, avian or mammalian host cellssuch as canine, feline, equine, bovine, ovine, or primate cellsincluding simian or human cells.

The filovirus genomic cDNA of the invention allows easy manipulation offilovirus, e.g., by the introduction of mutations into the viral genome.The methods of producing virus described herein, which do not requirehelper virus infection, are useful in viral mutagenesis studies, and inthe production of vaccines (e.g., for AIDS, influenza, hepatitis B,hepatitis C, rhinovirus, filoviruses, malaria, herpes, and foot andmouth disease) and gene therapy vectors (e.g., for cancer, AIDS,adenosine deaminase, muscular dystrophy, ornithine transcarbamylasedeficiency and central nervous system tumors). In particular, the use oflipid encapsulated particles of the invention which induce stronghumoral and cellular immunity may be preferred as vaccine vectors asthey are noninfectious and unlikely to give rise to infectiousrecombinant virus.

Thus, a virus for use in medical therapy (e.g., for a vaccine or genetherapy) is provided. For example, the invention provides a method toimmunize an animal against a pathogen, e.g., a bacteria, virus, orparasite, or a malignant tumor. The method comprises administering tothe animal an amount of at least one isolated virus of the inventionwhich encodes and expresses, or comprises, an immunogenic peptide orprotein of a pathogen or tumor, optionally in combination with anadjuvant, effective to immunize the animal. Alternatively, a lipidencapsulated particle of the invention may be used for immunization,either by delivering a DNA vaccine, or via expression of the immunogenicprotein on the surface of the particle, for instance, the particlecomprises a fusion protein comprising the extracellular domain of animmunogenic protein and the transmembrane and cytoplasmic portion offilovirus GP.

Also provided is a method to augment or increase the expression of anendogenous protein in an animal, e.g., a mammal such as a rodent,nonhuman primate or human, having an indication or disease characterizedby a decreased amount or a lack of the endogenous protein. The methodcomprises administering to the animal an amount of an isolated virus ofthe invention effective to augment or increase the amount of theendogenous protein in the animal. Alternatively, a lipid encapsulatedparticle of the invention can be employed to deliver DNA encoding theprotein or the protein itself. When the particle is used to deliverprotein, optionally the particle comprises a chimeric transmembraneprotein comprising an extracellular protein for targeting the particleto a specific tissue or cell type and the transmembrane and cytoplasmicportion of a filovirus GP.

The invention will be further described by the following non-limitingexamples.

Example 1 Generation of Transfectant Ebola Virus Materials and Methods

Efficiency of Virus Generation. To determine the efficiency of virusgeneration, Vero E6 cells were cotransfected with protein expressionplasmids and the plasmid for Ebola virus vRNA or cRNA synthesis. Fourdays after transfection, the efficiency of virus generation was measuredby determining the dose required to infect 50% of tissue culture cells(TCID₅₀) per ml of supernatant. The data shown in FIG. 1A arerepresentative results from three independent experiments. Experimentsfor the generation of Ebola virus as well as the characterization ofrecombinant Ebola virus were carried out in the BSL4 facility at theCanadian Science Centre for Human and Animal Health, Winnipeg, Canada.Cells were transfected with plasmids for the expression of the Ebolavirus NP, L, VP30, and VP35 proteins, and with the plasmid for Ebolavirus cRNA or vRNA synthesis, controlled by T7 RNA polymerase promoterand ribozyme sequences. T7 RNA polymerase was provided by cotransfectionof cells with pC-T7Pol.

Immunofluorescence Assay. Vero E6 cells were infected at a multiplicityof infection of 10⁻² with either wild-type Ebola virus generated fromplasmids or Ebola virus with an altered furin recognition sequence inits GP. Six days later, cells were observed under a light microscope.Three days after infection, cells were permeabilized and stained withantiserum. Three days after infection, cells were fixed with 2%paraformaldehyde in phosphate-buffered saline, followed by inactivationby gamma irradiation (2 Mrads). Cells were permeabilized with 0.1%Triton X-100 in phosphate-buffered saline for 15 minutes, washed threetimes with phosphate-buffered saline, and incubated for 1 hour at roomtemperature with an anti-Ebola virus Zaire rabbit antiserum (1:100dilution in phosphate-buffered saline). After three washes withphosphate-buffered saline, Cy3-labeled anti-rabbit (1:500) conjugate(Rockland, Gilbertsville, Pa.) was added for 1 hour at room temperature.The cells were then washed with phosphate-buffered saline, mounted, andanalyzed using an Axioplan 2 microscope (Zeiss).

Replication Kinetics. Vero E6 cells were infected with the wild-type orcleavage site mutant at a multiplicity of infection of 10⁻².Supernatants were harvested at 2, 24, 48, and 72 hours postinfection.The TCID₅₀ was determined by infecting Vero E6 cells with 10-folddilutions of the supernatants obtained at the above-mentioned timepoints.

Labeling of Protein and Immunoprecipitation Analysis. Vero E6 cells wereinfected at a multiplicity of infection of 10′ and incubated until acytopathic effect was observed. After the medium was removed, the cellswere washed once with methionine- and cysteine-free DMEM and labeled for24 hours in 2 ml of methionine- and cysteine-free Dulbecco's modifiedEagle's medium containing 2% dialyzed fetal calf serum and 10 μCi ofprotein labeling mix (NEN, Mississauga, Canada)/ml. The supernatantswere then clarified by centrifugation (1,000×g for 5 minutes at 4° C.).An equal volume of 2×RIPA buffer (2% Triton X-100, 2% sodiumdeoxycholate, 0.2% sodium dodecyl sulfate, 0.3 M NaCl, 40 mM Tris-HCl[pH 7.7], 20 mM EDTA [pH 8.0], 0.4 U of aprotinin/ml, 2 mMphenylmethylsulfonyl fluoride, 20 mM iodoacetamide) was added to thesupernatants, and the solutions were subsequently inactivated by gammairradiation (2 Mrad). Aliquots of the inactivated labeled material weremixed with an anti-Ebola virus Zaire horse serum and incubated at 4° C.overnight. The immune complexes were mixed with 30 μl of protein Gsepharose for 3 hours at 4° C. with rotation. After 3 washes with RIPAbuffer, the immunoprecipitated proteins were recovered by boiling themin 1×RIPA buffer.

Results

To generate a cDNA clone encoding the entire genome of Ebola virus(Zaire species, strain Mayinga), viral RNA was reverse transcribed withThermoScript Reverse Transcriptase (Gibco/BRL, Rockville, Md.) andamplified by PCR with Pfu Turbo (Stratagene, La Jolla, Calif.). Theresulting cDNA fragments were cloned in a Bluescript vector or itsderivatives. A consensus sequence was determined and compared to areference sequence (GenBank accession number AF086833). An A insertionwas found between nucleotides 9,744 and 9,745, which was also detectedin a partial Ebola virus genomic sequence (GenBank accession numberL11365). In addition, an A insertion was found between nucleotides18,495 and 18,496, and an A-to-T replacement was detected at position18,226. The latter two changes have also been reported for a functionalEbola virus minigenome (Muhlberger et al., 1999). A full-length Ebolavirus cDNA construct was assembled in a modified pTM1 vector (Moss etal., 1990), using conventional cloning techniques. Sequence analysis ofthe resulting full-length clone proved that no mutations had occurredduring cloning procedures in E. coli.

Negative-sense RNA viruses have been generated from constructs encodingeither the negative-sense viral RNA (vRNA) or the positive-sensecomplementary RNA (cRNA) (Marriott et al., 1999; Nagai et al., 1999;Neumann et al., 1999; Roberts et al., 1999; Schnell et al., 1994),albeit with higher efficiencies from the latter (Durbin et al., 1997;and Kato et al., 1996). cDNA constructs encoding the entire viral genomewere generated, flanked by the T7 RNA polymerase promoter and aribozyme, in both positive-sense and negative-sense orientations (FIG.1A). To achieve efficient transcription, the wild-type T7 RNA polymerasepromoter, which yields transcripts with an additional G at the 5′ end,was used to generate pTM-T7G-Ebo-Rib and pTM-Rib-Ebo-GT7 (FIG. 1A).

The generation of negative-sense RNA viruses requires viral proteins andgenomic RNA for replication and transcription. For Ebola virus, theproteins necessary for replication and transcription include theRNA-dependent RNA polymerase L, and the nucleoprotein (NP), and twoadditional auxiliary proteins (VP30 and VP35) (Muhlberger et al., 1999).To generate constructs for the expression of Ebola viral proteins, therespective cDNA fragments were amplified by PCR, the products sequencedand then cloned into the eukaryotic expression vector pCAGGS/MCS(controlled by the chicken β-actin promoter) (Kobasa et al., 1997; Niwaet al., 1991), resulting in four plasmids (pCEZ-NP, pCEZ-VP30,pCEZ-VP35, and pCEZ-L).

To generate Ebola virus, 5×10⁵ Vero E6 (African green monkey kidney)cells were transfected with 1 μg of the respective plasmid for Ebolavirus vRNA or cRNA synthesis and with the following amounts of proteinexpression plasmids: 1 μg of pCEZ-NP, 0.3 μg of pCEZ-VP30, 0.5 μg ofpCEZ-VP35, and 2 μg of pCEZ-L (FIG. 1B). To drive the transcription ofviral RNA from the T7 RNA polymerase promoter, cells were cotransfectedwith 1 μg of an expression plasmid for T7 RNA polymerase (pC-T7pol).Four days later, supernatants were collected and used to infect freshVero E6 cells. When examined at 6 to 8 days postinfection, the cellsshowed cytopathic effects, indicating the generation of infectious Ebolavirus entirely from cloned cDNA. Ebola virus was produced fromconstructs encoding either negative-sense vRNA or positive-sense cRNA(FIG. 1A). To determine the efficiency of virus generation, supernatantsof transfected cells were collected 4 days after transfection, and thetiter of virus in the supernatant was determined in Vero E6 cells. Theefficiencies of virus generation from negative-sense vRNA orpositive-sense cRNA were comparable, resulting in the generation of 10²50% tissue culture infective doses (TCID₅₀) per ml of supernatant.

A subsequent passage of the virus in Vero E6 cells was performed toconfirm the authenticity of the replicating agent. The first signs of acytopathic effect were observed at 48 hours postinfection and becamemore prominent during the following days (FIG. 2A). Indirectimmunofluorescence assays with a rabbit antiserum to Ebola virusGP/secreted GP (sGP) demonstrated the presence of Ebola virus GPs (FIG.2B). None of the negative controls (untreated cells or cells transfectedwith the full-length cDNA construct or the protein expression plasmidsalone) showed cytopathic effects or reacted with the anti-GP/sGPantiserum

The availability of a method for generating Ebola virus mutants greatlyincreases opportunities to dissect mechanisms of viral pathogenesis. Formany viruses, postranslational cleavage of membrane glycoproteins byhost proteolytic enzymes, including subtilisin-like proteases such asfurin, is a prerequisite for fusion between the viral envelope andcellular membranes and therefore an important step in pathogenesis(Klenk et al., 1994). The Ebola virus GP is cleaved by furin orfurin-like proteases at a highly conserved sequence motif (R—X—K/R—R; X,any amino acid) (Volchkov et al., 1998). Since the amino acid sequenceof the GP of the Reston species, the least pathogenic of all Ebola virussubtypes in humans, deviates from the optimal furin recognitionsequence, GP cleavage has been thought to be an important determinant ofEbola virus pathogenicity (Feldmann et al., 1999).

The effect of an altered furin recognition motif on Ebola virusreplication was studied by modifying pTM-T7G-Ebo-Rib. The multibasefurin recognition site (RRTRR at amino acid positions 497 to 501 of theGP) in pTM-T7G-Ebo-Rib was replaced with 497-AGTAA-501. The modifiedplasmid, designated pTM-T7G-Ebo-Rib-Cl(−), was transfected into Vero E6cells, together with protein expression plasmids for the NP, VP30, VP35,and L proteins and for T7 RNA polymerase. Fresh Vero E6 cells weresubsequently incubated with supernatants derived from the transfectedcells. Six days later, cytopathic effects were observed in these cells.Indirect immunofluorescence assays with antiserum to Zaire Ebola virusGP/sGP verified virus replication (FIG. 2). Growth curves in Vero E6cells demonstrated that although the mutant virus grew slightly moreslowly than the wild-type virus (FIG. 3), it reached 10¹⁰ TCID₅₀/ml at 3days postinfection.

To confirm the presence of mutations in the GP cleavage motif, wild-typeand mutant viruses were passaged three times in Vero E6 cells, RNAextracted from virions, and reverse transcriptase PCR performed withprimers spanning the altered furin recognition motif. Direct sequencingof the PCR products confirmed the retention of mutations in the GPcleavage site (data not shown).

FIG. 4 shows the results of experiments testing the cleavability of theEbola virus mutant GP lacking a furin recognition motif. Virions derivedfrom labeled Vero E6 cells infected with wild-type or mutant virus werelysed, and viral proteins were detected by immunoprecipitation using ahorse antiserum to Zaire Ebola virus. For wild-type virus, both cleavageproducts GP₁ (140 kDa) and GP₂ (26 kDa) were detected (FIGS. 4A and B,lanes 2). By contrast, alteration of the furin recognition sequenceabolished the generation of GP₁ and GP₂, and only the precursor, GP₀,was detected (FIGS. 4A and B, lanes 3), confirming that furin or relatedproteases are the major host cell proteases for GP cleavage. Theseresults indicate that the furin recognition motif at the Ebola GPcleavage site is dispensable for replication of the virus in cellculture.

Discussion

Marburg and Ebola viruses have been difficult to study because they mustbe handled in high-containment facilities, and effective methods ofexperimental mutagenesis were lacking. These limitations have restrictedthe development of antiviral drugs and vaccines, although reports ofpotentially useful experimental vaccines (Hevey et al., 1998; Sullivanet al., 2000; Vanderzanden et al., 1998; Xu et al., 1998) andantibody-mediated treatments (Maruyama et al., 1999; and Wilson et al.,2000) are beginning to emerge. The use of a reverse genetics system,which enables one to generate Ebola virus mutants entirely from clonedcDNA as described herein, opens a new era of filovirus research.

For many viruses in the Orthomyxoviridae and Paramyxoviridae families,GP cleavage by furin and other host cell proteases is absolutelyrequired for their infectivity and thus determines the extent of viralpathogenicity (Klenk et al., 1994). In contrast, findings with virusespseudotyped with Ebola GPs as well as the present results demonstratethat GP cleavage is dispensable for replication of Ebola virus, at leastin cell culture. The furin cleavage motif is highly conserved among allEbola GP sequences determined thus far, and its conservation suggests arole in the viral life cycle. Hence, GP cleavage by furin is notcritical for Ebola virus replication in the cells tested, but it may berequired for Ebola virus replication in vivo and/or in its naturalreservoir. Further studies with animal models will be needed toestablish the role of GP cleavage in Ebola virus replication andpathogenicity.

T7 RNA polymerase-based reverse genetics systems rely on the expressionof this enzyme within the transfected cells. To this end, two approacheshave been explored (reviewed in Marriott et al., 1999; Nagai et al.,1999; and Roberts et al., 1999). T7 RNA polymerase has been providedfrom recombinant vaccinia virus or from stable cell lines constitutivelyexpressing this enzyme. The former approach leaves investigators withthe task of separating the artificially generated recombinant virus fromvaccinia virus. On the other hand, cell lines expressing T7 RNApolymerase may produce insufficient amounts to efficiently transcribethe viral genome. In contrast to Volchkov et al. (2001), who used aBHK-21 cell line stably expressing T7 RNA polymerase, an entirelyplasmid-based system is described herein which was achieved using T7 RNApolymerase expression under control of the strong chicken β-actinpromoter. This approach resulted in 10² PFU of virus per ml of culturesupernatant. Expression of T7 RNA polymerase from plasmids may thereforebe an alternative for the generation of other nonsegmented,negative-sense RNA viruses, thereby circumventing restraints encounteredwith the established systems.

The reverse genetics systems for the generation of Ebola virus can beused to identify key regulatory elements and structure-functionrelationships in the viral life cycle, and allows the study ofmechanisms of filovirus pathogenicity in animal models. The system alsopromotes the development of new vaccines and the development ofreplication-deficient viruses.

Example 2 Generation of Noninfectious Ebola Particles Materials andMethods

Cells. 293 and 293T human embryonic kidney cells were maintained in DMEMsupplemented with 10% fetal calf serum, 2% L-glutamine, andpenicillin-streptomycin solution (DMEM-FCS) (Sigma). The cells weregrown at 37° C. in 5% CO₂.

Construction of Plasmids. To generate cDNA constructs encoding the VP40protein, primers were used that bind to the start and stop codons(positions 4479 and 5459 of the positive-sense antigenomic RNA) toreverse transcribe and PCR-amplify purified viral RNA (Titan RT-PCR Kit,Roche). The PCR product was cloned in the pT7Blue vector (Novagen)resulting in pT7EboZVP40. The cloned Ebola VP40 gene was sequenced toensure that unwanted nucleotide replacements were not present.

To generate plasmid pETEBoZVP40His for the expression of6-histidine-tagged VP40 in Escherichia coli, pT7EboZVP40 was used as atemplate for PCR amplification with the appropriate primers. The PCRproduct was blunt-end ligated into the SmaI-digested site of vector pM(CLONETECH). This construct was digested with NdeI and EcoRI and thefragment containing VP40 was ligated into the expression vector pET-5a(Promega). To generate plasmids pCEboZVP40, pCEboZVP40AAXY, pCEboZVP40M14A, pCEboZVP40/1-276, pCEboZVP40/1-226, pCEboZVP40/1-176,pCEboZVP40/50-326, and pCEboZVP40/100-326 (proteins expressed from theseplasmids are designated VP40, VP40AAXY, and the like) for expression ofVP40 and its mutants in eukaryotic cells, the Ebola Zaire VP40 gene wasamplified from pT7EboZVP40 using specific forward primers, eachcontaining an EcoRI site 5′ to the start of the coding region, andspecific reverse primers, each containing a BglII site 3′ to the stopcodon for each construct, and blunt-end ligated into the EcoRV-digestedsite of vector pT7Blue. Each construct was digested with EcoRI andBglII, and the fragment containing the VP40 gene or modified VP40 genewas cloned into the EcoRI and BglII-digested eukaryotic expressionvector pCAGGS/MCS (expression controlled by the chicken β-actinpromoter) (Kobasa et al., 1997; and Niwa et al., 1991). Eukaryoticexpression constructs employed in this study are schematically presentedin FIG. 5A.

Antibody. A polyclonal antibody against Ebola Zaire VP40 was produced asfollows: BL21 E. coli cells were transformed with plasmid pETEboZVP40His. Expression of the 6-His-tagged VP40 protein was induced with 1 mMIPTG for 3 hours. The E. coli cells were lysed and cellular debris wasremove by centrifugation. The supernatant was purified over an Ni-NTAagarose column (Qiagen). Expression of VP40 was verified by SDS-PAGEfollowed by Western blotting using a monoclonal antibody against thehistidine tag (Kodak). Rabbits were immunized with approximately 0.5 mgof VP40, and antibody against keratin present in the antiserum wasremoved with a keratin column (Girault et al., 1989).

Cell Transfection for Expression of VP40 and its Mutants. 293 or 293Tcells (60-mm plates) were transfected with expression vectors with theuse of the Trans IT LT-1 liposomal reagent (Panvera) according to themanufacturer's instructions. Briefly, DNA and transfection reagent weremixed (6 μl of Trans IT LT-1 with 3 μg of DNA) in 0.2 ml OPTI-MEM(Gibco-BRL), incubated for 30 minutes at room temperature, and added tothe cells. Transfected cells were incubated at 37° C. until harvest ofthe supernatant and/or cell monolayer.

Particle Formation Assay. Particles were assayed by the method of Li etal (1993) with some modifications. Forty-eight hours after transfectionof 293T cells with pCEboZVP40, pCEboZVP40AAXY, pCEboZVP40M14A, orpCEboZVP40/1-276, the culture medium was removed and placed on ice. Thecell monolayer was washed with phosphate-buffered saline (PBS), scrapedinto lysis buffer (0.25 M Tris-HCl, pH 8.0, 0.5% Triton X-100) and keptat 4° C. The culture medium (2 ml) was centrifuged at 2,000 rpm in amicrocentrifuge for 5 minutes to remove cellular debris, layered over20% sucrose in STE buffer (0.01 M Tris-Cl, pH 7.5, 0.01 M NaCl, 0.001 MEDTA, pH 8.0) (2 ml), and centrifuged at 150,000×g for 2 hours at 4° C.After centrifugation, the supernatant was removed and added to the celllysate. This mixture was saved for analysis of total protein expression.The pellet was resuspended in 1 ml STE buffer overnight at 4° C. Theresuspended pellet was layered over a 10-50% discontinuous sucrosegradient in STE buffer, centrifuged at 150,000×g for 4 hours at 4° C.,and fractions (1 ml) were collected from the top of the gradient. Eachfraction was mixed with 0.25 ml of 50% trichloroacetic acid (TCA) (10%TCA), the fractions were incubated for 30 minutes on ice, and theprecipitated proteins were pelleted by microcentrifugation for 15minutes. The pellets were washed once with cold acetone, air-dried, andresuspended in 0.05 ml SDS-PAGE sample buffer. Proteins in the mixtureof cell lysate and supernatant from centrifugation through 20% sucrosewere precipitated with 10% TCA, washed with acetone, and resuspended in0.5 ml SDS-PAGE sample buffer. Proteins were separated by 12% SDS-PAGEand detected by Western blotting. Fractions are numbered from the top tothe bottom of the gradient.

Protease Protection Assay. 293T cells were transfected with pCEboZVP40and, at 48 hours post-transfection, the culture medium was removed. Themedium was microcentrifuged at 2,000 rpm for 5 minutes to removecellular debris, layered over a 20% sucrose cushion, and centrifuged at165,000×g for 1 hour at 4° C. The supernatant was removed and the pelletwas resuspended overnight at 4° C. in 0.4 ml STE buffer. Thisresuspension was divided into six aliquots and treated following aprotocol previously described (Mik et al., 1989): Aliquot 1 received nofurther treatment; aliquot 2 was treated with soybean trypsin inhibitor(Biofluids) to a final concentration of 3 mg/ml; aliquot 3 with tritonX-100 to a final concentration of 1%; aliquot 4 with trypsin(Worthington) to a final concentration of 0.1 mg/ml; aliquot 5 with bothTriton X-100 to 1% and trypsin to 0.1 mg/ml final concentration; andaliquot 6 with both trypsin inhibitor (3 mg/ml final) and trypsin (0.1mg/ml final). The samples were incubated at room temperature for 30minutes, after which an excess of trypsin inhibitor (5 mg/ml) was addedto each aliquot. SDS-PAGE sample buffer (6X) was added to each aliquot.Proteins from each aliquot were separated by 12% SDS-PAGE and detectedby Western blotting.

Membrane-Association Assay. The method of Bergmann and Fusco (1988) wasused, with some modifications, to determine membrane-association of VP40and its mutants. Briefly, 48 hours after transfection of 293 cells withpCEboZVP40 or a mutant-VP40 expression plasmid, the culture medium wasremoved, and the cell monolayer, after a wash with (PBS), was scrapedinto ice-cold sucrose homogenization buffer (10% wt/wt sucrose, 10 mMTris-HCl (pH 7.4), 1 mM EDTA, and 10 mM iodoacetamide). Cells weredisrupted with 30 strokes of a Dounce homogenizer on ice andmicrocentrifuged for 3 minutes at 2,000 rpm to remove nuclei. Theresulting supernatant was made to 1 M NaCl or left untreated, incubatedat room temperature for 20 minutes, made to 80% sucrose (wt/vol), placedat the bottom of a Beckman SW41 centrifuge tube, and overlaid with 5 mlof 65% (wt/vol) sucrose and 2.5 ml of 10% sucrose. The gradient wascentrifuged to equilibrium at 150,000×g for 18 hours at 4° C. Fractions(1 ml) were collected from the top of the gradient, diluted 1:1 withTBS-Triton buffer (0.025 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% TritonX-100) or, for experiments involving expression of VP40/100-326,precipitated with TCA (as described for the particle formation assay)owing to the weak signal of this deletion construct in Western analysis,and mixed with SDS-PAGE sample buffer. Proteins from each aliquot wereseparated by 12% SDS-PAGE and detected by Western blotting.

Triton X-114 Phase Partitioning Analysis. The method used wasessentially that of Bordier (1981). Forty-eight hours post-transfectionof 293 cells pCEboZY40, pCEboZVP40/1-276, pCEboZVP40/1-226,pCEboZVP40/1-176, pCEboZP40/50-326, pCEboZVP40/100-326, or, as acontrol, a vector expressing A/WSN/33 (H₁N₁) influenza virushemagglutinin (HA), cells were scraped into cold TN buffer (10 mMTris-HCl, pH 7.4, 150 mM NaCl), disrupted with 30 strokes in a Douncehomogenizer, and subjected to centrifugation at 2,000 rpm for 3 minutesto remove nuclei. Triton X-114 (Sigma) was added to each supernatant to1%, and the resulting solution was incubated for 15 minutes at 4° C.with agitation. Unsolubilized material was pelleted by centrifugation ina picofuge for 5 minutes at 4° C., and the supernatant was heated to 37°C. for 5 minutes. The supernatant was layered onto a 37° C. sucrose (6%)cushion in TN buffer containing 0.06% Triton X-114 and centrifuged at2,000 rpm for 3 minutes at room temperature. The detergent (lower) andaqueous (upper) phases were recovered separately, the aqueous phase wasextracted a second time, like phases were pooled, and the detergentphase was diluted in TN buffer. Proteins in each phase were precipitatedwith 50% acetone and resuspended in SDS-PAGE sample buffer. Proteinswere separated by 12% SDS-PAGE and analyzed by Western blotting.

Western Blotting. Samples in sample buffer (10 μl) were incubated at100° C. for 5 minutes and separated on 12% polyacrylamide gels. Resolvedproteins were transferred to Westran polyvinylidine difluoride membranes(Schleicher & Schuell) and blocked overnight at 4° C. with 5% skim milkin PBST (0.05% Tween 20 (Sigma) in PBS). Blots were incubated in primaryantibody for 1 hour at room temperature, washed three times with PBST,incubated in biotinylated anti-rabbit secondary antibody (VectorLaboratories) for 30 minutes, washed three times with PBST, incubated instreptavidin-horseradish peroxidase reagent (Vector Laboratories) for 30minutes and washed three times with PBST. Blots were then incubated inLumi-Light Western blotting substrate (Boehringer-Mannheim) for 5minutes and exposed to x-ray film (Kodak).

Results

Expression of VP40 in Mammalian Cells. To ensure that VP40 is expressedat efficient levels in human embryonic kidney 293T cells, the celllysate was analyzed 24 hours after transfection with pCEboZVP40 byWestern blotting. Two bands reacting with anti-VP40 polyclonal antibodywere found, a small distance apart, in the range of 40 kDa (FIG. 6). Thelysate from cells transfected with the expression vector alone did notreact with the antibody.

VP40 contains an internal start codon at nucleotides 40-42 (codon 14)that is in frame with the first AUG. To determine whether proteinsynthesis from this internal start codon was responsible for thefaster-migrating band on the gel, a construct was generated,pCEboZVP40M14A, which expresses a mutant VP40 with this second AUGchanged to GCG, which encodes alanine, and expressed it as describedabove. Analysis of the cell lysate revealed a single, larger-sized band(FIG. 6), suggesting that the second AUG is used as a start codon to anappreciable extent in this system.

To determine whether loss of the PPXY motif at amino acids 10-13 of VP40affects expression of the protein, 293T cells were transfected withpCEboZVP40AAXY, which expresses a mutant VP40 in which the PPEY sequenceat amino acids 10-13 was changed to AAEY. Two bands corresponding tothose seen with the expression of wild-type VP40 were detected (FIG. 6).However, in contrast to the results obtained with wild-type VP40expression, where the slower-migrating band was the predominate product,pCEboZVP40AAXY expressed the two products at similar levels, indicatingthat loss of the PPXY motif affects either the translation of VP40 orits stability.

Production of Membrane-Bound Particles. To determine whetherVP40-associated vesicles are produced when the protein is expressed inthe absence of other viral proteins, 293T cells were transfected withpCEboZVP40 and, after 48 hours, collected the supernatant. After removalof cellular debris, the supernatant was subjected to ultracentrifugationover a 20% sucrose cushion. The pellet was resuspended and centrifugedthrough a 10-50% discontinuous sucrose gradient, and fractions wereanalyzed by Western blotting (FIG. 7). Fractions 6-8 contained VP40,with the majority of the protein found in fraction 7. The VP40 infractions 6-8 was most likely associated with membrane lipids in aparticle-like structure, as the sucrose densities in these fractionsranged from 1.11 to 1.13 g/ml, which corresponds to findings for matrixprotein-generated particles of other viruses (Giddings et al., 1998;Sandefur et al., 1998). Bands detected below full-length protein in thetotal protein fraction are likely degradation products. These dataindicate that VP40 expressed in the absence of other viral proteins canproduce membrane-bound particles.

Protease Protection Assay. To confirm the ability of VP40 to producemembrane-bound particles when expressed alone, a trypsin protectionassay was employed. Culture supernatant from cells transfected withpCEboZVP40 was centrifuged at 165,000×g through 20% sucrose, and thepellet was resuspended in STE buffer and divided into six equalaliquots. Aliquots 1-3 served as controls (untreated, trypsin inhibitortreated, and triton X-100 treated), aliquot 4 was treated with trypsin,aliquot 5 with trypsin and triton X-100, and aliquot 6 with trypsininhibitor and trypsin. Trypsin degraded VP40 only in the presence oftriton X-100 (FIG. 7), indicating that the viral protein does induce theproduction of fully membrane-bound particles; that is, trypsin digestionof VP40 required disruption of the lipid-bilayer surrounding theprotein.

VP40 Mutants and Membrane-Bound Particle Formation. Does the PPXY motifat amino acids 10-13 of VP40 contribute to particle production? Toaddress this question, VP40AAXY was expressed in 293T cells and assayedfor particles as described for wild-type VP40. VP40AAXY was not detectedin fractions corresponding to the sucrose densities to which wild-typeVP40 particles migrated (FIG. 7). Since VP40AAXY was synthesized atlevels similar to wild-type VP40, this finding indicates that mutationof the PPXY motif markedly disrupts VP40-generated vesicle formation.

FIG. 7 also shows the effect of loss of the second AUG codon on particleformation. A substantial amount of VP40M14A was present in fractions 5-8in the gradient, and the percentage of total VP40M14A expressed in 293Tcells that contributed to membrane-bound particle formation was muchgreater than the percentage of total wild-type VP40 involved in particleformation. This result is consistent with the finding that the PPXYmotif present immediately upstream of the second AUG is critical forVP40-associated particle formation (FIG. 7).

To determine whether the C-terminus of VP40 is essential for particleformation, a deletion mutant, VP40/1-276, was assayed which lacks thefinal 50 amino acids of VP40, for particle generation. Since thisdeletion mutant was not present at the same sucrose densities thatcharacterized the migration of wild-type VP40, it was concluded that thefirst 276 amino acids of VP40 are not sufficient for particle formation(FIG. 7).

VP40 Association with Cell Membranes and Structural Requirements forActivity. Flotation analysis was used to determine if VP40 bindscellular membranes efficiently in mammalian cells. In this method,postnuclear membrane fractions in 80% sucrose are loaded at the bottomof a centrifuge tube and overlaid with 65% and 10% sucrose. Duringcentrifugation, cellular membranes and their associated proteins floatto the 10-65% sucrose interface, while soluble proteins remain in thedense sucrose fractions at the bottom of the tube.

A large percentage of wild-type VP40 was found at the 10-65% sucroseinterface (fraction 3), while the remaining protein was found in theloading zone (fractions 8-12) (FIG. 9), indicating that VP40 does indeedbind cellular membranes. To clarify the interactions involved in thisassociation, VP40-associated membranes were treated with 1 M NaCl todetermine whether electrostatic interactions were required for thisassociation and subjected them to flotation analysis. Salt treatment hada negligible affect on the ability of VP40 to associate with membranes(FIG. 9), suggesting that the protein contains at least one hydrophobicdomain able to associate with membranes.

To elucidate the domain(s) of VP40 important for membrane association,deletion mutants were generated. Constructs expressing amino acids50-326 (pCEboZVP40/50-326), amino acids 100-326 (pCEboZVP40/100-326),amino acids 1-176 (pCEboZVP40/1-176), amino acids 1-226(pCEboZVP40/1-226), and amino acids 1-276 (pCEboZVP40/1-276) of VP40were expressed in 293 cells and their membrane association in thepresence or absence of 1 M NaCl was examined. The mutants with thelargest truncations, VP40/1-176 and VP40/100-326, showed the highestlevel of association with the lipid bilayer (FIG. 9). Salt treatment didnot affect these interactions. Mutants VP40/1-226 and VP40/50-326associated with membranes to the extent found with wild-type VP40, andthese interactions were also relatively unperturbed by treatment withsalt. By contrast, only a small portion of VP40/1-276 associated withthe lipid bilayer, and this interaction was eliminated upon treatmentwith salt. These results indicate that loss of the C-terminal 50 aminoacids of VP40 markedly alters the membrane-binding capabilities of VP40,primarily by disrupting hydrophobic interactions. This effect wasameliorated when 50 additional C-terminal amino acids were deleted, andmembrane-association was promoted when the protein was further truncatedto 176 amino acids. Deletion of the N-terminal 49 amino acids of VP40did not alter the membrane-binding characteristics of the protein,although truncation of 50 additional N-terminal amino acids did enhanceprotein-membrane association, as seen with VP40/1-176 (FIG. 9).

Since particle formation was markedly reduced with VP40AAXY, cellsexpressing this mutant were subjected to flotation analysis in order todetermine whether a decreased ability to bind membranes was involved inthis deficiency. As shown in FIG. 10, the loss of the PPXY motif in VP40did not affect the ability of the protein to bind membranes, indicatingthat lack of particle production with this mutant was not due to theloss of membrane association.

Flotation analysis was also used to determine whether the more efficientparticle formation induced by VP40M14A, by comparison to wild-type VP40,could be attributed, at least in part, to increased membrane binding bythis mutant. The percentage of VP40M14A associated with membranes wasonly slightly greater than that determined for wild-type VP40 (FIG. 10),indicating that this mutant relies on another mechanism to increaseparticle formation.

Triton X-114 Phase Partitioning Analysis. To probe the nature of theVP40-membrane interaction further, Triton X-114 phase partitioninganalysis was used as integral membrane proteins and lipid anchoredproteins partition in the detergent phase of a protein extraction andperipheral membrane proteins partition in the aqueous phase. FIG. 11shows the results of this analysis for wild-type VP40, the five deletionmutants of VP40, and influenza virus HA. HA, an integral membraneprotein, was found entirely in the detergent phase of the extraction, asexpected. Only a small portion of total VP40 was found in the detergentphase, while VP40/1-276 was found almost entirely in the aqueous phase.VP40/1-226 and VP40/50-326 partitioned in the detergent phase inproportions similar to that found for wild-type VP40. By contrast, whenVP40/1-176 and VP40/100-326 were expressed, large proportions of eachpartitioned in the detergent phase. These results indicate thatwild-type VP40 possesses only minor traits of an integral membraneprotein, and that deletion of its C-terminal 50 amino acids (VP40/1-276)abrogates these features. Further truncation of the C-terminus(VP40/1-226 and VP40/1-176) enhances the integral membrane character ofprotein. Deletion of the N-terminal 49 amino acids of VP40 (VP40/50-326)does not alter the general structural features of the protein, whiledeletion of amino acids 1-99 (VP40/100-326) appears to increase theextent of anchoring to lipids.

Discussion

Thus, VP40 of Ebola virus, when expressed in the absence of other viralproteins, can induce the formation of membrane-encompassed particles,much in the manner of the matrix proteins of VSV, rabies, and simianimmunodeficiency virus (Giddings et al., 1998; Harty et al., 1999;Justice et al., 1995; Li et al., 1993). Cellular proteins containing theWW domain are, in all likelihood, crucial for this process, as VP40containing an altered version of a PPXY motif at amino acids 10-13induces little or no particle formation. Harty et al. (1999)demonstrated that the matrix proteins of VSV and rabies viruses, whichpossess this motif at their N-termini, bind the cellularYes-kinase-associated and Nedd4 proteins via a PPXY motif-WW domain,interaction, and that the loss of this motif results in impaired virusrelease from infected cells. Jayakar et al. (2000) recently demonstratedthat mutation of the PPXY motif in the matrix protein of VSV impedesbudding of fully assembled virions at the plasma membrane. The datadescribed herein provides evidence for an important role of the PPXYmotif in particle formation induced by VP40, and suggest that cellularproteins are crucial players in this process.

The efficiency of particle production markedly increased when the secondATG codon of VP40 (codon 14) was changed to GCG (alanine), but thereason for this enhancement remains unclear. This ATG codon immediatelyfollows the PPXY motif. Perhaps the faster-migrating version of VP40,which lacks the PPXY motif, interferes with the assembly or budding offull-length VP40 molecules at the cell surface, or with the interactionbetween VP40 and a cellular protein. Whether translation from thissecond ATG occurs in actual viral infection or is an artifact of thesystem employed in this study is unknown.

Ruigrok et al. (2000) reported that VP40 expressed in E. coli can bindliposomes in vitro and that this interaction is largely electrostatic.In mammalian cells, a substantial amount of VP40 bound to the cellularmembrane, and that this interaction was disrupted negligibly by thepresence of 1 M NaCl, indicating that at least one hydrophobic domain isinvolved in this interaction. A small but appreciable portion of VP40partitioned with detergent in the manner of an integral membrane orlipid-anchored protein in Triton X-114 phase-partitioning analysis. Thisresult, together with the inability of 1 M NaCl to dissociate VP40 fromthe lipid bilayer, indicates that the protein has certain properties ofan integral membrane protein, as do a number of matrix proteins ofnegative-stranded RNA viruses (Chong et al., 1993; Zhang et al., 1996),even though Ebola VP40 does not appear to contain a region ofsignificant length and hydrophobicity to span the cell membrane (FIG.5B). Short hydrophobic stretches of VP40 may be able to penetrate thelipid bilayer to some extent, lending modest integral-membrane characterto the protein.

Ruigrok et al. (2000) also reported that a deletion mutant of VP40containing amino acids 31-212 failed to bind liposomes efficiently,indicating that the C-terminus of VP40 is absolutely required formembrane binding. To elucidate the domains involved in the associationof VP40 with cellular membranes, carboxy and amino-terminal deletionmutants were constructed. VP40 lacking its C-terminal 50 amino acidsdemonstrated appreciably reduced membrane association. TheKyte-Doolittle hydrophobicity plot (1982) of VP40 (FIG. 5B) indicatesthat amino acids 277-326 of the protein are primarily hydrophobic, sothat deletion of amino acids 277-326 eliminates a substantialhydrophobic region that is likely important for efficientmembrane-binding by the full-length protein. This hypothesis issupported by the fact that 1 M NaCl completely disrupted thisassociation, suggesting that affinity of this deletion construct withthe lipid bilayer depends primarily on electrostatic interactions.

When amino acids 227-326 of VP40 were deleted, the resulting truncatedprotein associated with the lipid bilayer as efficiently as wild-typeVP40; moreover, C-terminal deletion of amino acids 177-326 resulted in aprotein with much higher affinity for the lipid bilayer than was foundfor wild-type VP40. Salt treatment did not perturb membrane associationof these truncated versions of VP40, indicating the presence ofhydrophobic interactions mediated by the N-terminal 176 amino acids ofthe protein.

The hydrophobicity plot indicates that amino acids 227-276, andparticularly amino acids 177-226, are primarily hydrophilic. Deletion ofthe hydrophilic residues present in this region of VP40 may allow thetruncated protein to fold into a structure capable of strong hydrophobicassociation with the cell membrane, perhaps by effectively exposing thehighly hydrophobic central domain of the protein. These results areconsistent with data obtained by Triton X-114 extraction analysis (FIG.11). Since VP40 lacking its C-terminal 50 amino acids was unable toproduce particles (FIG. 7), and these C-terminal residues appear to berequired for efficient membrane association of VP40, binding of thishighly hydrophobic region to the lipid bilayer may be an essential stepin the particle formation process.

The crystal structure of amino acids 31-326 of Ebola virus was recentlyelucidated by Dessen et al. (2000). It shows VP40 to be distinct fromother viral matrix proteins, in that it consists of two similar domainsconnected by a flexible linker at amino acids 195-200. Ruigrok et al.(2000) showed that amino acids 31-212 of VP40 form hexamersspontaneously in solution. Dessen and associates postulate that, duringthe life cycle of Ebola virus, VP40 molecules associate with the lipidbilayer through interactions contributed primarily by their C-termini.After membrane binding, the molecules undergo a conformational changethat frees their N-termini for hexamerization. These hexamers then formbuilding blocks for a lattice that underlies the plasma membrane, andsubsequently may interact with the cytoplasmic tails of viralglycoproteins and/or the ribonucleoprotein complex. This model is basedon data demonstrating the hexamerization of VP40 molecules that lacktheir N-terminal 30 amino acids as well as their C-terminal 114 aminoacids. The PPXY motif that appears crucial for membrane-bound particleformation is located at amino acids 10-13 of VP40, and this motif mostlikely interacts with a cellular protein that exhibits a WW domainduring virus particle assembly or budding. It has not yet beendemonstrated that VP40 with a truncated C-terminus can form hexamerswhen the entire N-terminus is present. If hexamerization does occurduring virion morphogenesis, the 18 hexamers that form presumably mustleave the PPXY motif accessible to cellular proteins that participate inparticle formation and/or budding.

Example 3 Particles Comprising Filovirus Matrix Protein and GlycoproteinMaterials and Methods

Cells. 293T human embryonic kidney cells were maintained in Dulbecco'smodified Eagle medium supplemented with 10% fetal calf serum,L-glutamine and penicillin-streptomycin-gentamicin solution. The cellswere grown in an incubator at 37° C. in 5% CO₂.

Plasmids. Full-length cDNAs encoding the Ebola virus (species Zaire)VP40 or GP were cloned separately into a mammalian expression vector,pCAGGS/MCS (Kobasa et al., 1997; Niwa et al., 1991), which contains thechicken β-actin promoter. The resultant constructs were designatedpCEboZVP40 and pCEboZGP, respectively.

Cell Transfection for Expression of VP40 and GP. 293T cells (1×10⁶) weretransfected with plasmids using the Trans IT LT-1 reagent (Panvera,Madison, Wis.) according to the manufacturer's instructions. Briefly, 1μg of DNA in 0.1 ml Opti-MEM (Gibco-BRL) and 3 μl of the transfectionreagent were mixed, incubated for 10 minutes at room temperature, andadded to the cells. Transfected cells were incubated at 37° C. for 24 or48 hours.

Electron Microscopy. Ultrathin section electron microscopy was performedas follows. Twenty-four hours post-transfection of 293T cells withplasmids, the cells were washed with phosphate-buffered saline (PBS) andfixed for 20 minutes with 2.5% glutaraldehyde (GLA) in 0.1 M cacodylatebuffer (pH 7.4). They were scraped off the dish, pelleted by low-speedcentrifugation and then fixed for 30 minutes with the same fixative.Small pieces of fixed pellet were washed with the same buffer, postfixedwith 2% osmium tetroxide in the same buffer for 1 hour at 4° C.,dehydrated with a series of ethanol gradients followed by propyleneoxide, embedded in Epon 812 Resin mixture (TAAB) and polymerized at 70°C. for 2 days. For immune electron microscopy, cells were fixed with 4%paraformaldehyde and 0.1% GLA, dehydrated and embedded in LR White Resin(London Resin Company Ltd.). Thin sections were stained with uranilacetate and lead citrate, and examined with a JEM-1200EX electronmicroscope at 80 Kv.

For negative staining, culture media of 293T cells were collected at 24hours post-transfection onto a Formvar-coated copper grid, stained with2% phosphotungstic acid solution (PTA) and examined with a JEM-1200electron microscope at 80 Kv.

For immune electron microscopy, the samples were absorbed toFormvar-coated nickel grids and washed with PBS containing 0.5% bovineserum albumin (PBS-BSA). The grids were then treated with mouse anti-GPmonoclonal antibody (a mixture of ZGP12, ZGP42, and ZGP133 (31); 1:150in PBS-BSA) or rabbit anti-VP40 polyclonal antibody (1:300 in PBS-BSA),and rinsed six times with PBS, followed by incubation with a goatantimouse immunoglobulin conjugated to 15-nm gold particles (1:50dilution; BBlnternational) or a goat antirabbit immunoglobulinconjugated to 5-nm gold particles (1:100 dilution; BBlnternational).After washing, the samples were fixed for 10 min in 2% glutaraldehydeand negatively stained with 2% PTA.

Results

Pleomorphic Particle Formation by GP. To determine the morphology ofvesicles induced by Ebola virus GP expression, GP-expressing cells andtheir supernatants were analyzed by electron microscopy. The ultrathinsections of these cells showed particle-like structures with surfacespikes budding from the plasma membrane (FIG. 12A); no such structureswere observed using cells transfected with the expression vector alone(FIG. 12B). As previously observed in the recombinant vaccinia virussystem (Volchkov et al., 1998), pleomorphic structures similar tovirosomes with a range of diameters were apparent in the supernatants ofGP-expressing cells (FIGS. 13 A and B). The spikes on the surface of thevesicles reacted with anti-GP monoclonal antibodies (FIGS. 13 C and D),confirming the GP derivation of the structures.

VP40 Induces Filamentous Particle Formation. To determine how VP40protein expressed in 293T cells is released into culture medium (Hartyet al., 2000; Timmins et al., 2001; Example 2), the VP40-expressingcells were analyzed by transmission electron microscopy. The ultrathinsections of the cells expressing VP40 showed budding of filamentousstructures (approximately 65 nm in diameter) on the cell surface (FIG.14A). In some cells, the plasma membranes appeared ruffled and toconsist of two bilayers (FIG. 14C). Aggregated ribosomes (FIG. 14E,arrows) were occasionally found in the cytoplasm of cells expressingVP40, as were electron-dense filamentous structures (approximately 45 nmin diameter; FIG. 14F, arrowheads), which were never seen in cellstransfected with the expression vector alone. The budding particles andmembrane ruffles reacted with rabbit anti-VP40 polyclonal antibody(FIGS. 14B and D), confirming that VP40 had contributed to thegeneration of these structures. In studies to further determine the sizeand morphology of the VP40 particles released from cells, thesupernatants of cells expressing this protein were centrifuged through20% sucrose, and the pelleted material was negatively stained with 2%PTA and analyzed by electron microscopy. Filamentous particles, whichhad uniform diameters of approximately 65 nm but varied lengths, wereobserved (FIGS. 15A-C). These results indicate that VP40 alone caninduce the formation of filamentous particles, which bud from the cellsurface.

VP40-GP Interaction in Particle Morphogenesis. To determine how GPexpression affects VP40-driven particle formation, 293T cells weretransfected with both VP40- and GP-expressing plasmids. In ultrathinsections of the transfected cells, filamentous particle-like structuresof 80-nm external diameter were observed that were budding from theplasma membrane (FIGS. 16A and B). The structures possessed spikes ofapproximately 10 nm on their surface, in contrast to the structuresobserved in cells expressing VP40 alone (FIG. 14A). Also, unlike thefindings with expression of GP alone, few pleomorphic particles wereobserved. The particle structures were studied in more detail afternegative staining of the particles in culture supernatants of cellsexpressing both VP40 and GP. Filamentous Ebola virus-like particles withsurface spikes of approximately 85-nm in external diameter and lengthsthat ranged to 10 μm were observed (FIGS. 17A-C). The spikes projectedfrom the particle surface at 5- to 10-nm intervals and weremorphologically indistinguishable from those on the Ebola virion surface(Feldmann et al., 1996; Peters et al., 1995). Labeling of the spikeswith a mixture of anti-GP monoclonal antibodies conjugated with goldparticles confirmed their identity as GP (FIG. 17D). Furthermore, whentreated with 0.03% Triton X-100 and with both the anti-VP40 antibodyconjugated to 5-nm gold particles and a mixture of anti-GP monoclonalantibodies conjugated to 15-nm gold particles, the filamentous particlesbecame labeled with both antibodies, demonstrating that the Ebolavires-like particles contained GP as well as VP40 proteins (FIG. 17E).These results demonstrate GP incorporation into VP40-generatedfilamentous structures, without affecting filamentous particleformation.

Discussion

A hallmark of Ebola virus is its filamentous virions as featured in itsfamily name Filoviridae. The shape of enveloped viruses are determinedby viral proteins in retroviruses (Campbell et al., 1997; Gay et al.,1998; Joshi et al., 2000) or by both viral RNA length and proteins inVSV (Pattnaik et al., 1991). Because specific interactions among viralcomponents are required for the formation of defined virion shapes,understanding of such interactions can lead to the identification oftargets for the development of antiviral compounds.

As shown herein by electron microscopy, the expression of VP40 in theabsence of any other Ebola vires proteins leads to the formation offilamentous particles, which resemble spikeless virions released intothe supernatant of cultured Ebola virus-infected cells (Geisbert et al.,1995). Thus, these results suggest that the Ebola virus VP40 possessesstructural information necessary and sufficient to induce the formationof filamentous particles, which then bud from the plasma membrane.Interestingly, some filamentous structures were observed in thecytoplasm of cells expressing VP40 as have been found in the cytoplasmof the cells infected with Ebola virus. Similar structures have alsobeen observed in cells expressing the M1 protein of influenza virus orthe GAG protein of retrovirus (Delchambre et al., 1989; Gheyson et al.,1989; Gomez-Puertas et al., 2000). However, the tubular structuresobserved upon expression of influenza virus M1 alone were not seenduring normal viral infection or when M1 was coexpressed with otherinfluenza viral proteins. Thus, VP40 may form intracellular filamentousstructures by self-aggregation.

Membrane ruffles containing VP40 protein were observed in someVP40-expressing cells (FIGS. 14C and D). The M protein of VSV inducessimilar double-layered membranes at the cell surface when expressed fromrecombinant Sendai virus (Sakaguchi et al, 1999). IpaC protein secretedby Shigella flexneri has also been linked to large-scale membraneextension in macrophages, including lamellipodia and membrane ruffles(Kuwae et al, 2001; Tran Van Nhieu et al., 1999), while Salmonellatyphimurium triggers the formation of host cell membrane ruffles innonphagocytic cells (Ginocchio et al., 1994; Zhou et al., 1999). Thesemembrane ruffles are thought to result from interactions between thebacterial proteins, including IpaC, and the actin cytoskeletons of hostcells (Tran Van Nhieu et al., 1999; Zhou et al., 1999). In Ebolavirus-infected cells, host cell plasma membranes proliferate extensivelyat the peak stage of viral budding (Geisbert et al, 1995), as observedin cells expressing VP40 alone. Thus, VP40 may interact with actinfilaments during the assembly or budding of Ebola virus at the cellsurface.

The impact of glycoprotein interaction with the matrix protein on virionmorphology differs among viruses. For example, deletion of thecytoplasmic tails of the influenza virus hemagglutinin and neuraminidasealters virus morphology (Jin et al., 1997; Mitnaul et al., 1996), whilethe characteristic morphology of rabies virus and VSV do not depend onglycoprotein-matrix protein interaction (Mebatsion et al, 1996;Mebatsion et al., 1994; Schnell et al., 1998; Takada et al., 1997). TheEbola virus GP, like VSV-G, was incorporated into filamentous particleswithout affecting the morphology of the particles. However, suchinteraction may contribute to the efficiency of budding, as demonstratedby research on VSV (Jayakar et al., 2000; Mebatsion et al., 1999).

In conclusion, VP40 induces VP40 containing-filamentous particleformation and GP spikes are incorporated into VP40 induced-filamentousparticles upon coexpression of GP and VP40, resulting in Ebolavirus-like particles. This virus-like particle formation system will beuseful to further elucidate the mechanism of Ebola virus particleformation, including the functional link among Ebola viral and cellularcomponents.

REFERENCES

-   Baize et al., Nat. Med., 5, 423 (1999).-   Basler et al., Proc. Natl. Acad. Sci. U.S.A., 97, 12289 (2000).-   Bergmann et al., J. Cell Biol., 107, 1707 (1988).-   Bordier, J. Biol. Chem., 256, 1604 (1981).-   Bork et al., Trends Biochem. Sci., 19, 531 (1994).-   Campbell et al., J. Virol., 71, 4425 (1997).-   Chen et al., Proc. Natl. Acad. Sci. U.S.A., 92, 7819 (1995).-   Chong et al., J. Virol., 67, 407 (1993).-   Coronel et al., J. Virol., 73, 7035 (1999).-   Delchambre et al., EMBO. J., 8, 2653 (1989).-   Dessen et al., EMBO J., 19, 4228 (2000).-   Durbin et al., Virology, 235, 323 (1997).-   Elliott et al., Virology, 147, 169 (1985).-   Feldmann et al., Arch. Virol. Suppl., 15, 159 (1999).-   Feldmann et al., Arch. Virol. Suppl., 7, 81 (1993).-   Feldmann et al., 1996. Marburg and Ebola viruses. P. 1-52. In    Maramorosch, K., Murphy, F. A. and Shatkin, A. J. (ed.), Advances in    virus research 47, Academic Press.-   Feldmann et al., Filoviruses, p. 651-664, 9^(th) ed. Edward Arnold,    London, United Kingdom (1998).-   Garoff et al., Microbiol. Mol. Biol. Rev., 62, 1171 (1998).-   Gay et al., Virology, 247, 160 (1998).-   Geisbert et al., Virus Res., 39, 129 (1995).-   Gheysen et al., Cell, 59, 103 (1989).-   Giddings et al., Virology, 248, 108 (1998).-   Ginocchio et al., Cell, 76, 717 (1994).-   Girault et al., Anal. Biochem., 182, 193 (1989).-   Gomez-Puertas et al., J. Virol., 74, 11538 (2000).-   Haffer et al., J. Virol., 64, 2653 (1990).-   Harty et al., J. Virol., 73, 2921 (1999).-   Harty et al., Proc. Natl. Acad. Sci. U.S.A., 97, 13871 (2000).-   Hevey et al., Virology, 251, 28 (1998).-   Ito et al., J. Virol., 75, 1576 (2001).-   Jasenosky et al., J. Virol., 75, 5205 (2001).-   Jayakar et al., J. Virol., 74, 9818 (2000).-   Jin et al., EMBO. J., 16, 1236 (1997).-   Joshi et al., J. Virol., 74, 10260 (2000).-   Justice et al., J. Virol., 69, 3156 (1995).-   Kato et al., Genes Cells, 1, 569 (1996).-   Klenk et al., Trends Microbiol., 2, 39 (1994).-   Kobasa et al., J. Virol., 71, 6706 (1997).-   Kuwae et al., J. Biol. Chem., 276, 32230 (2001).-   Kyte et al., J. Mol. Biol., 157, 105 (1982).-   Li et al., J. Virol., 67, 4415 (1993).-   Marriott et al., Adv. Virus Res., 53, 321 (1999).-   Maruyama et al., J. Virol., 73, 6024 (1999).-   Mebatsion et al., Cell, 84, 941 (1996).-   Mebatsion et al., J. Virol., 73, 242 (1999).-   Mitnaul et al., J. Virol., 70, 873 (1996).-   Moss et al., Nature, 348, 91 (1990).-   Moss, Poxyiridae: The viruses and their replication. P. 2637-2672.    In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields    virology, Lippincott-Raven Publishers, Philadelphia, Pa. 1996.-   Muhlberger et al., J. Virol., 73, 2333 (1999).-   Nagai et al., Microbiol. Immunol., 43, 613 (1999).-   Neumann et al., J. Virol., 74, 547 (2000).-   Neumann et al., Proc. Natl. Acad. Sci. USA, 96, 9345 (1999).-   Niwa et al., Gene, 108, 193 (1991).-   Pattnaik et al., Proc. Natl. Acad. Sci. USA, 88, 1379 (1991).-   Peters et al., 1995. Filoviridae: Marburg and Ebola viruses. P.    1161-1176. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.),    Fields virology, Lippincott-Raven Publishers, Philadelphia, Pa.-   Roberts et al., Adv. Virus Res., 53, 301 (1999).-   Ruigrok et al., J. Mol. Biol., 300, 103 (2000).-   Sakaguchi et al., Virology, 263, 230 (1999).-   Sanchez et al., Proc. Natl. Acad. Sci. U.S.A., 93, 3602 (1996).-   Sanchez et al., Virus Res., 29, 215 (1993).-   Sandefur et al., J. Virol., 72, 2723 (1998).-   Schnell et al., EMBO J., 13, 4195 (1994).-   Schnell et al., EMBO. J., 17, 1289 (1998).-   Sudol et al., FEBS Lett., 369, 67 (1995).-   Sullivan et al., Nature, 408, 605 (2000).-   Takada et al., J. Virol., 75, 2324 (2001).-   Takada et al., Proc. Natl. Acad. Sci. U.S.A., 94, 14764 (1997).-   Takimoto et al., J. Virol., 75, 11384 (2001).-   Timmins et al., Virology, 283, 1 (2001).-   Tran Van Nhieu et al., EMBO. J., 18, 3249 (1999).-   Vanderzanden et al., Virology, 246, 134 (1998).-   Volchkov et al., J. Gen. Virol., 80, 355 (1999).-   Volchkov et al., Proc. Natl. Acad. Sci. USA, 95, 5762 (1998).-   Volchkov et al., Science, 291, 1965 (2001).-   Volchkov et al., Virology, 245, 110 (1998).-   Wills et al., J. Virol., 63, 4331 (1989).-   Wilson et al., Science, 287, 1664 (2000).-   Wilson et al., Science, 287, 1664 (2000).-   Wool-Lewis et al., J. Virol., 73, 1419 (1999).-   Wool-Lewis, et al., J. Virol. 72, 3155 (1998).-   Xu et al., Nat. Med., 5, 373 (1998).-   Yang et al., Nat. Med., 6, 886 (2000).-   Yang et al., Science, 279, 1034 (1998).-   Zhang et al., Virology, 225, 255 (1996).-   Zhou et al., Proc. Natl. Acad. Sci. USA, 96, 10176 (1999).

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1. A method to prepare filovirus, comprising: contacting a cell with avector comprising a promoter operably linked to a filovirus genomic cDNAor a portion thereof linked to a transcription termination sequence, avector comprising a promoter operably linked to a DNA segment encoding afilovirus RNA transcriptase-polymerase, a vector comprising a promoteroperably linked to a DNA segment encoding filovirus NP, a vectorcomprising a promoter operably linked to a DNA segment encodingfilovirus VP30, and a vector comprising a promoter operably linked to aDNA segment encoding filovirus VP35, so as to yield infectiousfilovirus, wherein the portion of the cDNA, when transcribed, yields aRNA which is capable of being packaged into filovirus virions or whichis capable of being replicated in the presence of filovirus proteins. 2.The method of claim 1 wherein the promoter in the vector comprisingfilovirus genomic cDNA is a RNA polymerase I promoter, RNA polymerase IIpromoter, RNA polymerase III promoter, T7 RNA polymerase promoter, or T3RNA polymerase promoter.
 3. The method of claim 1 further comprising avector comprising a promoter operably linked to a DNA fragment ofinterest.
 4. The method of claim 1 wherein the vector comprising thefilovirus genomic cDNA further comprises a DNA fragment of interestwithin the genomic sequence.
 5. The method of claim 1 wherein thepromoter of the vector comprising the filovirus genomic cDNA is a T7 RNApolymerase promoter.
 6. The method of claim 5 further comprising avector comprising a promoter operably linked to a DNA segment encodingT7 RNA polymerase.
 7. The method of claim 1 further comprising isolatingthe virus.
 8. The method of claim 3 or 4 wherein the DNA fragment ofinterest encodes a detectable marker, a therapeutic protein or animmunogenic polypeptide or peptide of a pathogen or tumor antigen. 9.The method of claim 1 wherein the sequence of the genomic cDNA has oneor more nucleotide deletions, insertions or substitutions relative tothe sequence of a corresponding wild-type filovirus.
 10. A compositioncomprising a plurality of filovirus vectors, comprising: a) a vectorcomprising a promoter operably linked to a filovirus genomic cDNA or aportion thereof linked to a transcription termination sequence, whereinthe portion of the cDNA, when transcribed, yields a RNA which is capableof being packaged into filovirus virions or which is capable of beingreplicated in the presence of filovirus proteins; and b) a vectorcomprising a promoter operably linked to a DNA segment encoding afilovirus RNA transcriptase-polymerase, a vector comprising a promoteroperably linked to a DNA segment encoding filovirus NP, a vectorcomprising a promoter operably linked to a DNA segment encodingfilovirus VP30, and a vector comprising a promoter operably linked to aDNA segment encoding filovirus VP35.
 11. The composition of claim 10further comprising a vector comprising a promoter operably linked to aDNA fragment of interest.
 12. The composition of claim 10 wherein thevector of a) further comprises a DNA fragment of interest in the sameorientation as the genomic cDNA.
 13. The composition of claim 11 or 12wherein the DNA fragment of interest encodes an immunogenic polypeptideor peptide of a pathogen, a tumor antigen, or a therapeutic protein. 14.The composition of claim 10 wherein each vector of b) is on a separateplasmid.
 15. The composition of claim 10 further comprising a vectorcomprising a promoter operably linked to a DNA segment encoding T7 RNApolymerase, wherein the promoter of the vector of a) is a T7 RNApolymerase promoter.
 16. The composition of claim 10 wherein each of thevectors of b) further comprise a transcription termination sequence. 17.The composition of claim 10 wherein the cDNA is in the senseorientation.
 18. The composition of claim 10 wherein the cDNA is in theantisense orientation.
 19. A cell contacted with the composition ofclaim
 10. 20. The cell of claim 19 which is a eukaryotic cell.
 21. Thecell of claim 19 which is an insect, yeast, or mammalian cell.
 22. Avector encoding a mutant filovirus matrix protein having one or moreamino acid deletions, insertions or substitutions relative to wild-typefilovirus matrix protein, which mutant binds to lipid.
 23. The vector ofclaim 22 which encodes a mutant filovirus matrix protein having residuescorresponding to residues 1-276, 1-226, 50-276, 50-326 or 100-326 of theEbola virus matrix protein.
 24. The vector of claim 22 wherein themutant filovirus matrix protein binds to the cell membrane of eukaryoticcells.